LAUSR

Ubiquitin‐conjugating (E2) enzymes in protein ubiquitination are associated with various diseases. An artificial RING finger (ARF) is a useful tool, and E2 activities are conveniently estimated based on ARF reactivities. To extend the use of ARF in cells, we constructed a TAT‐ARF using a cell‐penetrating trans‐activator protein (TAT) peptide. An in vitro ubiquitination assay without substrates showed auto‐ubiquitination of TAT‐ARF via its TAT region. TAT‐ARF was translocated into MCF7 breast cancer cells, and then TAT‐ARF ubiquitinated itself via its ARF. Experiments using confocal laser‐scanning microscopy revealed that FAM‐labeled TAT‐ARF was readily internalized in cells and it remained encapsulated in vesicles. The Cell Counting Kit‐8 assay indicated that the TAT‐ARF uptake occurred without cytotoxicity in MCF7 cells at concentrations below 5.0 μM. By taking advantage of TAT‐ARF, we, for the first time, succeeded in detecting E2 activities in cells. Thus, the present work opens up new avenues in the investigation of protein ubiquitination.